Methods for Treating Bleeding Disorders Using Sulfated Polysaccharides

ABSTRACT

Methods for treating bleeding disorders using non-anticoagulant sulfated polysaccharides (NASPs) as procoagulants are disclosed. NASPs can be administered as single agents, or in combination with one another, or, with other medications (such as factors VII, VIII and IX) to promote hemostasis. In particular, the use of NASPs in treatment of bleeding disorders, including congenital coagulation disorders, acquired coagulation disorders, and trauma induced hemorrhagic conditions is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/140,504, filed May 27, 2005, which claims the benefit under 35 U.S.C.§119(e)(1) of U.S. provisional application 60/574,845, filed May 27,2004, which applications are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

This invention relates to the treatment of bleeding disorders, includingcongenital coagulation disorders, acquired coagulation disorders, andtrauma induced hemorrhagic conditions. In particular, this inventionrelates to the use of non-anticoagulant sulfated polysaccharides (NASP)to improve clotting and hemostasis in hemophilic conditions.

BACKGROUND

Normal blood coagulation is a complex physiological and biochemicalprocess involving activation of a coagulation factor cascade leading tofibrin formation and platelet aggregation along with localvasoconstriction (reviewed by Davie et al., Biochemistry 30:10363,1991). The clotting cascade is composed of an “extrinsic” pathwaythought to be the primary means of normal coagulation initiation and an“intrinsic” pathway contributing to an expanded'coagulation response.The normal response to a bleeding insult involves activation of theextrinsic pathway. Activation of the extrinsic pathway initiates whenblood comes in contact with tissue factor (TF), a cofactor for factorVII that becomes exposed or expressed on tissues following insult. TFforms a complex with FVII that facilitates the production of FVIIa.FVIIa then associates with TF to convert FX to the serine protease FXa,which is a critical component of the prothrombinase complex. Theconversion of prothrombin to thrombin by theFXa/FVa/calcium/phospholipid complex stimulates the formation of fibrinand activation of platelets, all of which is essential to normal bloodclotting. Normal hemostasis is further enhanced by intrinsic pathwayfactors IXa and VIIIa, which also convert FX to FXa.

Blood clotting is inadequate in bleeding disorders, which may be causedby congenital coagulation disorders, acquired coagulation disorders, orhemorrhagic conditions induced by trauma. Bleeding is one of the mostserious and significant manifestations of disease, and may occur from alocal site or be generalized. Localized bleeding may be associated withlesions and may be further complicated by a defective haemostaticmechanism. Congenital or acquired deficiencies of any of the coagulationfactors may be associated with a hemorrhagic tendency. Congenitalcoagulation disorders include hemophilia, a recessive X-linked disorderinvolving a deficiency of coagulation factor VIII (hemophilia A) orfactor IX (hemophilia B) and von Willebrands disease, a rare bleedingdisorder involving a severe deficiency of von Willebrands factor.Acquired coagulation disorders may arise in individuals without aprevious history of bleeding as a result of a disease process. Forexample, acquired coagulation disorders may be caused by inhibitors orautoimmunity against blood coagulation factors, such as factor VIII, vonWillebrand factor, factors IX, V, XI, XII and XIII; or by hemostaticdisorders such as caused by liver disease, which may be associated withdecreased synthesis of coagulation factors. Coagulation factordeficiencies are typically treated by factor replacement which isexpensive, inconvenient (intravenous), and not always effective. As manyas 20% of patients receiving chronic factor replacement therapy maygenerate neutralizing antibodies to replacement factors.

Thus, there remains a need for new therapeutic approaches for treatingbleeding disorders. A single pharmaceutical agent that is safe,convenient and effective in a broad range of bleeding disorders wouldfavorably impact clinical practice.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for treatingbleeding disorders using non-anticoagulant sulfated polysaccharides(NASPs) as procoagulants. NASPs can be administered as single agents, orin combination with one another, or with other hemostatic agents. Inparticular, the use of NASPs in treatment of bleeding disorders,including congenital coagulation disorders, acquired coagulationdisorders, and trauma induced hemorrhagic conditions is described.

In one aspect, the invention provides a method for treating a subject inneed of enhanced blood coagulation comprising administering atherapeutically effective amount of a composition comprising anon-anticoagulant sulfated polysaccharide (NASP) to the subject. Incertain embodiments, the invention provides a method for treating asubject having a bleeding disorder comprising administering atherapeutically effective amount of a composition comprising a NASP tothe subject. In certain embodiments, the NASP is selected from the groupconsisting of N-acetyl-heparin (NAH), N-acetyl-de-O-sulfated-heparin(NA-de-o-SH), de-N-sulfated-heparin (De-NSH),de-N-sulfated-acetylated-heparin (De-NSAH), periodate-oxidized heparin(POH), chemically sulfated laminarin (CSL), chemically sulfated alginicacid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS),heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS), andfucoidan.

In other embodiments the NASP is selected from the group consisting oflow molecular weight fragments of the previously listed compounds. Inpreferred embodiments the fragment of the NASP decreases blood clottingtime when tested in the dPT assay. In one embodiment, the NASP is afragment of fucoidan that decreases blood clotting time when tested inthe dPT assay.

In further embodiments, the NASP can be coadministered with one or moredifferent NASPs and/or in combination with one or more other therapeuticagents.

In certain embodiments, a NASP is administered to a subject to treat ableeding disorder selected from the group consisting of hemophilia A,hemophilia B, von Willebrand disease, idiopathic thrombocytopenia, adeficiency of one or more contact factors, such as Factor XI, FactorXII, prekallikrein, and high molecular weight kininogen (HMWK), adeficiency of one or more factors associated with clinically significantbleeding, such as Factor V, Factor VII, Factor VIII, Factor IX, FactorX, Factor XIII, Factor II (hypoprothrombinemia), and von Willebrandsfactor, a vitamin K deficiency, a disorder of fibrinogen, includingafibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia, analpha₂-antiplasmin deficiency, and excessive bleeding such as caused byliver disease, renal disease, thrombocytopenia, platelet dysfunction,hematomas, internal hemorrhage, hemarthroses, surgery, trauma,hypothermia, menstruation, and pregnancy.

In certain embodiments, a NASP is administered to a subject to treat acongenital coagulation disorder or an acquired coagulation disordercaused by a blood factor deficiency. The blood factor deficiency may becaused by deficiencies of one or more factors, including but not limitedto, factor V, factor VII, factor VIII, factor IX, factor XI, factor XII,factor XIII, and von Willebrand factor.

In certain embodiments, the subject having a bleeding disorder isadministered a therapeutically effective amount of a compositioncomprising a NASP in combination with another therapeutic agent. Forexample, the subject may be administered a therapeutically effectiveamount of a composition comprising a NASP and one or more factorsselected from the group consisting of factor XI, factor XII,prekallikrein, high molecular weight kininogen (HMWK), factor V, factorVII, factor VIII, factor IX, factor X, factor XIII, factor II, factorVIIa, and von Willebrands factor. Treatment may further compriseadministering a procoagulant such as thrombin; an activator of theintrinsic coagulation pathway, including factor Xa, factor IXa, factorXIa, factor XIIa, and VIIIa, prekallekrein, and high-molecular weightkininogen; or an activator of the extrinsic coagulation pathway,including tissue factor, factor VIIa, factor Va, and factor Xa.Therapeutic agents used to treat a subject having a bleeding disordercan be administered in the same or different compositions andconcurrently, before, or after administration of a NASP.

In another aspect, the invention provides a method for reversing theeffects of an anticoagulant in a subject, the method comprisingadministering a therapeutically effective amount of a compositioncomprising a non-anticoagulant sulfated polysaccharide (NASP) to thesubject. In certain embodiments, the subject may have been treated withan anticoagulant including, but not limited to, heparin, a coumarinderivative, such as warfarin or dicumarol, tissue factor pathwayinhibitor (TFPI), antithrombin III, lupus anticoagulant, nematodeanticoagulant peptide (NAPc2), active-site blocked factor VIIa (factorVIIai), factor IXa inhibitors, factor Xa inhibitors, includingfondaparinux, idraparinux, DX-9065a, and razaxaban (DPC906), inhibitorsof factors Va and VIIIa, including activated protein C (APC) and solublethrombomodulin, thrombin inhibitors, including hirudin, bivalirudin,argatroban, and ximelagatran. In certain embodiments, the anticoagulantin the subject may be an antibody that binds a clotting factor,including but not limited to, an antibody that binds to Factor V, FactorVII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II, FactorXI, Factor XII, von Willebrands factor, prekallikrein, or high molecularweight kininogen (HMWK).

In certain embodiments, a NASP can be coadministered with one or moredifferent NASPs and/or in combination with one or more other therapeuticagents for reversing the effects of an anticoagulant in a subject. Forexample, the subject may be administered a therapeutically effectiveamount of a composition comprising a NASP and one or more factorsselected from the group consisting of factor XI, factor XII,prekallikrein, high molecular weight kininogen (HMWK), factor V, factorVII, factor VIII, factor IX, factor X, factor XIII, factor II, factorVIIa, and von Willebrands factor. Treatment may further compriseadministering a procoagulant, such as an activator of the intrinsiccoagulation pathway, including factor Xa, factor IXa, factor XIa, factorXIIa, and VIIIa, prekallekrein, and high-molecular weight kininogen; oran activator of the extrinsic coagulation pathway, including tissuefactor, factor VIIa, factor Va, and factor Xa. Therapeutic agents usedin combination with a NASP to reverse the effects of an anticoagulant ina subject can be administered in the same or different compositions andconcurrently, before, or after administration of the NASP.

In another aspect, the invention provides a method for treating asubject undergoing a surgical or invasive procedure wherein improvedblood clotting would be desirable, comprising administering atherapeutically effective amount of a composition comprising anon-anticoagulant sulfated polysaccharide (NASP) to the subject. Incertain embodiments, the NASP can be coadministered with one or moredifferent NASPs and/or in combination with one or more other therapeuticagents to the subject undergoing a surgical or invasive procedure. Forexample, the subject may be administered a therapeutically effectiveamount of one or more factors selected from the group consisting offactor XI, factor XII, prekallikrein, high molecular weight kininogen(HMWK), factor V, factor VII, factor VIII, factor IX, factor X, factorXIII, factor II, factor VIIa, and von Willebrands factor. Treatment mayfurther comprise administering a procoagulant, such as an activator ofthe intrinsic coagulation pathway, including factor Xa, factor IXa,factor XIa, factor XIIa, and VIIIa, prekallekrein, and high-molecularweight kininogen; or an activator of the extrinsic coagulation pathway,including tissue factor, factor VIIa, factor Va, and factor Xa.Therapeutic agents used to treat a subject undergoing a surgical orinvasive procedure can be administered in the same or differentcompositions and concurrently, before, or after administration of theNASP.

In another aspect, the invention provides a method of inhibiting TFPIactivity in a subject, the method comprising administering atherapeutically effective amount of a composition comprising a NASP tothe subject.

In another aspect, the invention provides a method of inhibiting TFPIactivity in a biological sample, the method comprising combining thebiological sample (e.g., blood or plasma) with a sufficient amount of anon-anticoagulant sulfated polysaccharide (NASP) to inhibit TFPIactivity.

In another aspect, the invention provides a composition comprising aNASP. In certain embodiments, the NASP is selected from the groupconsisting of N-acetyl-heparin (NAH), N-acetyl-de-O-sulfated-heparin(NA-de-o-SH), de-N-sulfated-heparin (De-NSH),de-N-sulfated-acetylated-heparin (De-NSAH), periodate-oxidized heparin(POH), chemically sulfated laminarin (CSL), chemically sulfated alginicacid (CSAA), chemically sulfated pectin (CSP), dextran sulfate (DXS),heparin-derived oligosaccharides (HDO), pentosan polysulfate (PPS), andfucoidan. In other embodiments the NASP is selected from the groupconsisting of low molecular weight fragments of the previously listedcompounds. In certain embodiments, the composition may further comprisea pharmaceutically acceptable excipient. In certain embodiments, thecomposition may further comprise one or more different NASPs, and/or oneor more therapeutic agents, and/or reagents. For example, thecomposition may further comprise one or more factors selected from thegroup consisting of factor XI, factor XII, prekallikrein, high molecularweight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX,factor X, factor XIII, factor II, and von Willebrands factor, tissuefactor, factor VIIa, factor Va, and factor Xa, factor IXa, factor XIa,factor XIIa, and VIIIa; and/or one or more reagents selected from thegroup consisting of APTT reagent, thromboplastin, fibrin, TFPI,Russell's viper venom, micronized silica particles, ellagic acid,sulfatides, and kaolin.

In another aspect, the invention provides a method of measuringacceleration of clotting by a NASP in a biological sample, the methodcomprising:

-   -   a) combining the biological sample with a composition comprising        the NASP,    -   b) measuring the clotting time of the biological sample,    -   c) comparing the clotting time of the biological sample to the        clotting time of a corresponding biological sample not exposed        to the NASP, wherein a decrease in the clotting time of the        biological sample exposed to the NASP, if observed, is        indicative of a NASP that accelerates clotting.

In certain embodiments, one or more different NASPs and/or therapeuticagents, and/or reagents can be added to the biological sample formeasurements of clotting time. For example, one or more factors can beadded, including but not limited to, factor XI, factor XII,prekallikrein, high molecular weight kininogen (HMWK), factor V, factorVII, factor VIII, factor IX, factor X, factor XIII, factor II, and vonWillebrands factor, tissue factor, factor VIIa, factor Va, and factorXa, factor IXa, factor XIa, factor XIIa, and VIIIa; and/or one or morereagents, including but not limited to, APTT reagent, tissue factor,thromboplastin, fibrin, TFPI, Russell's viper venom, micronized silicaparticles, ellagic acid, sulfatides, and kaolin.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the increase in clotting time of hemophilia A (Hem-A)plasma in the presence of tissue factor pathway inhibitor (TFPI)determined by the dPT assay. A plot of clotting time (seconds) versusTFPI concentration (μg/ml) shows that clotting time increases linearlywith TFPI dose.

FIG. 2 compares anticoagulant activities of potential NASPs,N-acetyl-heparin (NAH), N-acetyl-de-O-sulfated-heparin (NA-de-O-SH),de-N-sulfated-heparin (De-N-SH), de-N-sulfated-acetylated-heparin(De-N-SAH), pentosan polysulfate (PPS), fucoidan, and heparin. Selectedpolysaccharides were tested at various concentrations in Hem-A plasma.FIG. 2 shows a plot of clotting time (seconds) versus NASP concentration(nM). Data points shown are mean values from duplicate measurements.

FIG. 3 compares the effects of NAH, PPS, fucoidan, and heparin onclotting time of Hem-A plasma containing 1.25% FACT plasma, asdetermined using the aPTT assay. FIG. 3 shows a plot of clotting time(seconds) versus NASP concentration (nM). Data points shown are meanvalues from duplicate measurements.

FIG. 4 shows that NASPs, including NAH, PPS, and fucoidan accelerateclotting of Hem-A plasma containing recombinant TFPI. NASPs were brieflypreincubated with TFPI prior to addition to plasma. Clotting times weredetermined using the dPT assay. A plot of clotting time (seconds) versusNASP concentration (nM) is shown. Data points shown are mean values fromduplicate measurements. NASP inhibition of TFPI activity resulted inreduced plasma clotting times.

FIG. 5 shows that NASPs, including NAH, PPS, and fucoidan accelerateclotting of hemophilia B (Hem-B) plasma containing recombinant TFPI.NASPs were briefly preincubated with TFPI prior to addition to plasma.Clotting times were determined using the dPT assay. A plot of clottingtime (seconds) versus NASP concentration (nM) is shown. Data pointsshown are mean values from duplicate measurements. NASP inhibition ofTFPI activity resulted in reduced plasma clotting times.

FIG. 6 shows that NAH, PPS, and fucoidan accelerate clotting of Hem-Aplasma containing TFPI without preincubation of TFPI with NASPs prior tointroduction of TFPI into plasma. A plot of clotting time (seconds)versus NASP concentration (nM) is shown. Clotting times were determinedusing the dPT assay. Data points shown are mean values from duplicatemeasurements.

FIG. 7 shows that PPS and fucoidan accelerate clotting of Hem-A plasmain the absence of exogenous TFPI supplementation. The dose-response ofNASPs is compared to a positive control, factor VIIa, for amplificationof extrinsic pathway activation. FIG. 7 shows a plot of clotting time(seconds) versus NASP concentration (nM). Clotting times were determinedusing the dPT assay. Data points shown are mean values from duplicatemeasurements.

FIG. 8 shows that fucoidan and PPS accelerate clotting of factorVII-deficient plasma in dPT assays. Clotting time was measured followingpreincubation of factor VII-deficient plasma with varying concentrationsof fucoidan or PPS. FIG. 8 shows a plot of clotting time (seconds)versus NASP concentration (nM). Data points shown are mean values fromduplicate measurements.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of pharmacology, chemistry,biochemistry, coagulation, recombinant DNA techniques and immunology,within the skill of the art. Such techniques are explained fully in theliterature. See, e.g., Handbook of Experimental Immunology, Vols. I-IV(D. M. Weir and C. C. Blackwell eds., Blackwell ScientificPublications); A. L. Lehninger, Biochemistry (Worth Publishers, Inc.,current addition); Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N.Kaplan eds., Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in theirentireties.

I. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a NASP” includes a mixture of two or more such agents, andthe like.

A “NASP” as used herein refers to a sulfated polysaccharide thatexhibits anticoagulant activity in a dilute prothrombin time (dPT) oractivated partial thromboplastin time (aPTT) clotting assay that is nomore than one-third, and preferably less than one-tenth, the molaranticoagulant (statistically significant increase in clotting time)activity of unfractionated heparin (MW range 8,000 to 30,000; mean18,000 daltons). NASPs may be purified and/or modified from naturalsources (e.g. brown algae, tree bark, animal tissue) or may besynthesized de novo and may range in molecular weight from 100 daltonsto 1,000,000 daltons. NASPs may be used in the methods of the inventionfor improving hemostasis in treating bleeding disorders, particularlythose associated with deficiencies of coagulation factors or forreversing the effects of anticoagulants. The ability of NASPs to promoteclotting and reduce bleeding is readily determined using various invitro clotting assays (e.g., dPT and aPTT assays) and in vivo bleedingmodels (e.g. tail snip, transverse cut, whole blood clotting time, orcuticle bleeding time determination in hemophilic mice or dogs). See,e.g., PDR Staff. Physicians' Desk Reference. 2004, Anderson et al.(1976) Thromb. Res. 9:575-580; Nordfang et al. (1991) Thromb Haemost.66:464-467; Welsch et al. (1991) Thrombosis Research 64:213-222; Brozeet al. (2001) Thromb Haemost 85:747-748; Scallan et al. (2003) Blood.102:2031-2037; Pijnappels et al. (1986) Thromb. Haemost. 55:70-73; andGiles et al. (1982) Blood 60:727-730.

A “procoagulant” as used herein refers to any factor or reagent capableof initiating or accelerating clot formation. A procoagulant of theinvention includes any activator of the intrinsic or extrinsiccoagulation pathways, such as a clotting factor selected from the groupconsisting of factor Xa, factor IXa, factor XIa, factor XIIa, and VIIIa,prekallekrein, high-molecular weight kininogen, tissue factor, factorVIIa, and factor Va. Other reagents that promote clotting includekallikrein, APTT initiator (i.e., a reagent containing a phospholipidand a contact activator), Russel's viper venom (RVV time), andthromboplastin (for dPT). Contact activators that can be used in themethods of the invention as procoagulant reagents include micronizedsilica particles, ellagic acid, sulfatides, kaolin or the like known tothose of skill in the art. Procoagulants may be from a crude naturalextract, a blood or plasma sample, isolated and substantially purified,synthetic, or recombinant. Procoagulants may include naturally occurringclotting factors or fragments, variants or covalently modifiedderivatives thereof that retain biological activity (i.e., promoteclotting). Optimal concentrations of the procoagulant can be determinedby those of skill in the art.

The term “polysaccharide,” as used herein, refers to a polymercomprising a plurality (i.e., two or more) of covalently linkedsaccharide residues. Linkages may be natural or unnatural. Naturallinkages include, for example, glycosidic bonds, while unnaturallinkages may include, for example, ester, amide, or oxime linkingmoieties. Polysaccharides may have any of a wide range of averagemolecular weight (MW) values, but generally are of at least about 100daltons. For example, the polysaccharides can have molecular weights ofat least about 500, 1000, 2000, 4000, 6000, 8000, 10,000, 20,000,30,000, 50,000, 100,000, 500,000 daltons or even higher. Polysaccharidesmay have straight chain or branched structures. Polysaccharides mayinclude fragments of polysaccharides generated by degradation (e.g.,hydrolysis) of larger polysaccharides. Degradation can be achieved byany of a variety of means known to those skilled in the art includingtreatment of polysaccharides with acid, base, heat, or enzymes to yielddegraded polysaccharides. Polysaccharides may be chemically altered andmay have modifications, including but not limited to, sulfation,polysulfation, esterification, and methylation.

The term “derived from” is used herein to identify the original sourceof a molecule but is not meant to limit the method by which the moleculeis made which can be, for example, by chemical synthesis or recombinantmeans.

The terms “variant,” “analog” and “mutein” refer to biologically activederivatives of the reference molecule, that retain desired activity,such as clotting activity in the treatment of a bleeding disorderdescribed herein. In general, the terms “variant” and “analog” inreference to a polypeptide (e.g., clotting factor) refer to compoundshaving a native polypeptide sequence and structure with one or moreamino acid additions, substitutions (generally conservative in nature)and/or deletions, relative to the native molecule, so long as themodifications do not destroy biological activity and which are“substantially homologous” to the reference molecule as defined below.In general, the amino acid sequences of such analogs will have a highdegree of sequence homology to the reference sequence, e.g., amino acidsequence homology of more than 50%, generally more than 60%-70%, evenmore particularly 80%-85% or more, such as at least 90%-95% or more,when the two sequences are aligned. Often, the analogs will include thesame number of amino acids but will include substitutions, as explainedherein. The term “mutein” further includes polypeptides having one ormore amino acid-like molecules including but not limited to compoundscomprising only amino and/or imino molecules, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), polypeptides with substituted linkages, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring (e.g., synthetic), cyclized, branched moleculesand the like. The term also includes molecules comprising one or moreN-substituted glycine residues (a “peptoid”) and other synthetic aminoacids or peptides. (See, e.g., U.S. Pat. Nos. 5,831,005; 5,877,278; and5,977,301; Nguyen et al., Chem. Biol. (2000) 7:463-473; and Simon etal., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions ofpeptoids). Preferably, the analog or mutein has at least the sameclotting activity as the native molecule. Methods for making polypeptideanalogs and muteins are known in the art and are described furtherbelow.

As explained above, analogs generally include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

By “derivative” is intended any suitable modification of the referencemolecule of interest or of an analog thereof, such as sulfation,acetylation, glycosylation, phosphorylation, polymer conjugation (suchas with polyethylene glycol), or other addition of foreign moieties, solong as the desired biological activity (e.g., clotting activity,inhibition of TFPI activity) of the reference molecule is retained. Forexample, polysaccharides may be derivatized with one or more organic orinorganic groups. Examples include polysaccharides substituted in atleast one hydroxyl group with another moiety (e.g., a sulfate, carboxyl,phosphate, amino, nitrile, halo, silyl, amido, acyl, aliphatic,aromatic, or a saccharide group), or where a ring oxygen has beenreplaced by sulfur, nitrogen, a methylene group, etc. Polysaccharidesmay be chemically altered, for example, to improve procoagulantfunction. Such modifications may include, but are not limited to,sulfation, polysulfation, esterification, and methylation. Methods formaking analogs and derivatives are generally available in the art.

By “fragment” is intended a molecule consisting of only a part of theintact full-length sequence and structure. A fragment of apolysaccharide may be generated by degradation (e.g., hydrolysis) of alarger polysaccharide. Active fragments of a polysaccharide willgenerally include at least about 2-20 saccharide units of thefull-length polysaccharide, preferably at least about 5-10 saccharideunits of the full-length molecule, or any integer between 2 saccharideunits and the full-length molecule, provided that the fragment inquestion retains biological activity, such as clotting activity and/orthe ability to inhibit TFPI activity. A fragment of a polypeptide caninclude a C-terminal deletion an N-terminal deletion, and/or an internaldeletion of the native polypeptide. Active fragments of a particularprotein will generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, or any integer between 5 amino acids andthe full-length sequence, provided that the fragment in question retainsbiological activity, such as clotting activity, as defined herein.

“Substantially purified” generally refers to isolation of a substance(e.g., sulfated polysaccharide) such that the substance comprises themajority percent of the sample in which it resides. Typically in asample a substantially purified component comprises 50%, preferably80%-85%, more preferably 90-95% of the sample. Techniques for purifyingpolysaccharides, polynucleotides, and polypeptides of interest arewell-known in the art and include, for example, ion-exchangechromatography, affinity chromatography and sedimentation according todensity.

By “isolated” is meant, when referring to a polysaccharide orpolypeptide, that the indicated molecule is separate and discrete fromthe whole organism with which the molecule is found in nature or ispresent in the substantial absence of other biological macro-moleculesof the same type.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two nucleic acid, or two polypeptide sequencesare “substantially homologous” to each other when the sequences exhibitat least about 50%, preferably at least about 75%, more preferably atleast about 80%-85%, preferably at least about 90%, and most preferablyat least about 95%-98% sequence identity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified sequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two molecules(the reference sequence and a sequence with unknown % identity to thereference sequence) by aligning the sequences, counting the exact numberof matches between the two aligned sequences, dividing by the length ofthe reference sequence, and multiplying the result by 100. Readilyavailable computer programs can be used to aid in the analysis, such asALIGN; Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O.Dayhoff ed., 5 Suppl. 3:353-358, National biomedical ResearchFoundation, Washington, D.C., which adapts the local homology algorithmof Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 forpeptide analysis. Programs for determining nucleotide sequence identityare available in the Wisconsin. Sequence Analysis Package, Version 8(available from Genetics Computer Group, Madison, Wis.) for example, theBESTFIT, FASTA and GAP programs, which also rely on the Smith andWaterman algorithm. These programs are readily utilized with the defaultparameters recommended by the manufacturer and described in theWisconsin Sequence Analysis Package referred to above. For example,percent identity of a particular nucleotide sequence to a referencesequence can be determined using the homology algorithm of Smith andWaterman with a default scoring table and a gap penalty of sixnucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs are readily available.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

By “vertebrate subject” is meant any member of the subphylum chordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The invention described herein is intended for use in any ofthe above vertebrate species.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aNASP of the invention, and includes both humans and animals.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, including but not limited to, forexample, blood, plasma, serum, fecal matter, urine, bone marrow, bile,spinal fluid, lymph fluid, samples of the skin, external secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, organs, biopsies and also samples of in vitrocell culture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

By “therapeutically effective dose or amount” of a NASP, blood factor,or other therapeutic agent is intended an amount that, when administeredas described herein, brings about a positive therapeutic response, suchas reduced bleeding or shorter clotting times.

The term “bleeding disorder” as used herein refers to any disorderassociated with excessive bleeding, such as a congenital coagulationdisorder, an acquired coagulation disorder, or a trauma inducedhemorrhagic condition. Such bleeding disorders include, but are notlimited to, hemophilia A, hemophilia B, von Willebrand disease,idiopathic thrombocytopenia, a deficiency of one or more contactfactors, such as Factor XI, Factor XII, prekallikrein, and highmolecular weight kininogen (HMWK), a deficiency of one or more factorsassociated with clinically significant bleeding, such as Factor V,Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II(hypoprothrombinemia), and von Willebrands factor, a vitamin Kdeficiency, a disorder of fibrinogen, including afibrinogenemia,hypofibrinogenemia, and dysfibrinogenemia, an alpha₂-antiplasmindeficiency, and excessive bleeding such as caused by liver disease,renal disease, thrombocytopenia, platelet dysfunction, hematomas,internal hemorrhage, hemarthroses, surgery, trauma, hypothermia,menstruation, and pregnancy.

II. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

A. General Overview

Blood clotting disorders including hemophilia (Hem) A and Hem B, severevon Willebrand disease (svWD), and severe factor VII (FVII) deficiencyhave typically been treated by factor replacement, e.g., factor VIII forHem A and svWD, factor IX for Hem B, and factor VII(a) forFVII-deficiency and others (recently reviewed in Bishop et al. (2004)Nat. Rev. Drug Discov. 3:684-694; Carcao et al. (2004) Blood Rev.18:101-113; Roberts et al. (2004) Anesthesiology 100:722-730; and Lee(2004) Int. Anesthesiol. Clin. 42:59-76). While such therapies are ofteneffective, characteristics limiting utility include high cost,inconvenience (i.e. intravenous administration), and neutralizingantibody generation (Bishop et al., supra; Carcao et al., supra; Robertset al., supra; Lee, supra; and Bohn et al. (2004) Haemophilia 10 Suppl.1:2-8). While FVIIa is increasingly utilized in various bleedingdisorders (Roberts et al., supra), alternative single compoundprocoagulant therapies devoid of aforementioned constraints and withbroad application are of interest.

One general approach to improving hemostasis in individuals withbleeding disorders is to improve the initiation of clotting byupregulating the extrinsic pathway of blood coagulation. While theintrinsic and extrinsic pathways of coagulation contribute to thrombingeneration and fibrin clot formation (Davie et al. (1991) Biochemistry30:10363-10370), the extrinsic—or tissue factor (TF) mediated—path iscritical for initiation, and contributes to propagation of coagulationin vivo (Mann (2003) Chest 124(3 Suppl):1S-3S; Rapaport et al. (1995)Thromb. Haemost. 74:7-17). One potential mechanism for upregulatingextrinsic pathway activity is the attenuation of Tissue Factor PathwayInhibitor (TFPI). TFPI is a Kunitz-type proteinase inhibitor of FVIIa/TFthat provides tonic downregulation of extrinsic pathway activation (seeBroze (1992) Semin. Hematol. 29:159-169; Broze (2003) J. Thromb.Haemost. 1:1671-1675; and Johnson et al. (1998) Coron. Artery Dis.9(2-3):83-87 for review). Indeed, heterozygous TFPI deficiency in micecan result in exacerbation of thrombus formation (Westrick et al. (2001)Circulation 103:3044-3046), and TFPI gene mutation is a risk factor forthrombosis in humans (Kleesiek et al. (1999) Thromb. Haemost. 82:1-5).Regulating clotting in hemophilia via the targeting of TFPI wasdescribed by Nordfang et al. and Wun et al., who showed that anti-TFPIantibodies could shorten the coagulation time of hemophilic plasma(Nordfang et al. (1991) Thromb. Haemost. 66:464-467; Welsch et al.(1991) Thromb. Res. 64:213-222) and that anti-TFPI IgG improved thebleeding time of rabbits that were factor VIII-deficient (Erhardtsen etal. (1995) Blood Coagul. Fibrinolysis 6:388-394).

As a class, sulfated polysaccharides are characterized by a plethora ofbiological activities with often favorable tolerability profiles inanimals and humans. These polyanionic molecules are often derived fromplant and animal tissues and encompass a broad range of subclassesincluding heparins, glycosaminoglycans, fucoidans, carrageenans,pentosan polysulfates, and dermatan or dextran sulfates (Toida et al.(2003) Trends in Glycoscience and Glycotechnology 15:29-46). Lowermolecular weight, less heterogeneous, and chemically synthesizedsulfated polysaccharides have been reported and have reached variousstages of drug development (Sinay (1999) Nature 398:377-378; Bates etal. (1998) Coron. Artery Dis. 9:65-74; Orgueira et al. (2003) Chemistry9:140-169; McAuliffe (1997) Chemical Industry Magazine 3:170-174;Williams et al. (1998) Gen. Pharmacol. 30:337-341). Heparin-likesulfated polysaccharides exhibit differential anticoagulant activitymediated through antithrombin III and/or heparin cofactor IIinteractions (Toida et al., supra). Notably, certain compounds, ofnatural origin or chemically modified, exhibit other biologicalactivities at concentrations (or doses) at which anticoagulant activityis not substantial (Williams et al. 1998) Gen. Pharmacol. 30:337-341;Wan et al. (2002) Inflamm. Res. 51:435-443; Bourin et al. (1993)Biochem. J. 289 (Pt 2):313-330; McCaffrey et al. (1992) Biochem.Biophys. Res. Commun. 184:773-781; Luyt et al. (2003) J. Pharmacol. Exp.Ther. 305:24-30). In addition, heparin sulfate has been shown to exhibitstrong interactions with TFPI (Broze (1992) Semin. Hematol. 29:159-169;Broze (2003) J. Thromb. Haemost. 1:1671-1675; Johnson et al. (1998)Coron. Artery Dis. 9:83-87; Novotny et al. (1991) Blood; 78(2):394-400):

As described herein, certain sulfated polysaccharides interact with TFPIand inhibit its activity at lower concentrations than those associatedwith anticoagulation. Such molecules may be of use in settings whereclot formation is compromised.

B. NASPs as Promoters of Clotting

The present invention is based on the discovery that non-anticoagulantsulfated polysaccharides (NASPs) can be used as procoagulants intreatment of patients with bleeding disorders. A novel approach forregulating hemostasis has been discovered by the inventors herein that,paradoxically, utilizes sulfated polysaccharides, such as heparin-likesulfated polysaccharides to promote clotting. Selected sulfatedpolysaccharides described herein are largely devoid of anticoagulantactivity, or exhibit clot-promoting activity at concentrationssignificantly lower than the concentration at which they exhibitanticoagulant activity, and are hence denoted “non-anticoagulantsulfated polysaccharides.”

As shown in Examples 4-6, NASPs promote clotting of plasma from subjectsthat have hemophilia A (Hem-A) or hemophilia B (Hem-B) according todilute prothrombin time (dPT) and activated partial thromboplastin time(aPTT) clotting assays. In addition, NASPs reduce bleeding time inhemophilia A and B mouse models following injury (Example 7). In theexperiments disclosed herein, certain candidate NASPs are shown inclotting assays to demonstrate at least ten-fold lower anticoagulantactivity as compared to heparin. Moreover, a subset of NASPs, includingpentosan polysulfate (PPS) and fucoidan, inhibited Tissue Factor PathwayInhibitor (TFPI) and improved (i.e. accelerated) the clotting time ofhuman hemophilia A and hemophilia B plasmas or plasma with reduced FVIIlevels when tested at concentrations ranging from 4-500 nM in diluteprothrombin time (dPT) assays. Improved hemostasis in vivo was observedin mice with hemophilia A or B following low dose subcutaneousadministration of PPS or fucoidan, or a combination of NASP and a factorsupplement. Increased survival of factor deficient mice following ableeding challenge was also observed. These results indicate thatsystemic administration of select NASPs may represent a unique approachfor regulating hemostasis in bleeding disorders.

Thus, the invention relates to the use of NASPs to control hemostasis insubjects with bleeding disorders, including congenital coagulationdisorders, acquired coagulation disorders, and trauma inducedhemorrhagic conditions.

C. NASPs

NASPs for use in the methods of the invention are sulfatedpolysaccharides that have procoagulant activity. The noncoagulantproperties of potential NASPs are determined using dilute prothrombintime (dPT) or activated partial thromboplastin time (aPTT) clottingassays. Noncoagulant sulfated polysaccharides exhibit no more thanone-third, and preferably less than one-tenth, the anticoagulantactivity (measured by statistically significant increase in clottingtime) of unfractionated heparin (MW range 8,000 to 30,000; mean 18,000daltons).

Sulfated polysaccharides with potential NASP activity include, but arenot limited to, glycosaminoglycans (GAGs), heparin-like moleculesincluding N-acetyl heparin (Sigma-Aldrich, St. Louis, Mo.) andN-desulfated heparin (Sigma-Aldrich), sulfatoids, polysulfatedoligosaccharides (Karst et al. (2003) Cum Med. Chem. 10:1993-2031;Kuszmann et al. (2004) Pharmazie. 59:344-348), chondroitin sulfates(Sigma-Aldrich), dermatan sulfate (Celsus Laboratories Cincinnati,Ohio), fucoidan (Sigma-Aldrich), pentosan polysulfate (PPS)(Ortho-McNeil Pharmaceuticals, Raritan, N.J.), fucopyranon sulfates(Katzman et al. (1973) J. Biol. Chem. 248:50-55), and novel sulfatoidssuch as GM1474 (Williams et al. (1998) General Pharmacology 30:337) andSR 80258A (Burg et al. (1997) Laboratory Investigation 76:505), andnovel heparinoids, and their analogs. NASPs may be purified and/ormodified from natural sources (e.g. brown algae, tree bark, animaltissue) or may be synthesized de novo and may range in molecular weightfrom 100 daltons to 1,000,000 daltons. Additional compounds withpotential NASP activity include periodate-oxidized heparin (POH)(Neoparin, Inc., San Leandro, Calif.), chemically sulfated laminarin(CSL) (Sigma-Aldrich), chemically sulfated alginic acid (CSAA)(Sigma-Aldrich), chemically sulfated pectin (CSP) (Sigma-Aldrich),dextran sulfate (DXS) (Sigma-Aldrich), heparin-derived oligosaccharides(HDO) (Neoparin, Inc., San Leandro, Calif.).

In principle, any free hydroxyl group on a monosaccharide component of aglycoconjugate can be modified by sulfation to produce a sulfatedglycoconjugate for potential use as a NASP in the practice of theinvention. For example, such sulfated glycoconjugates may includewithout limitation sulfated mucopolysaccharides (D-glucosamine andD-glucoronic acid residues), curdlan (carboxymethyl ether, hydrogensulfate, carboxymethylated curdlan) (Sigma-Aldrich), sulfatedschizophyllan (Itoh et al. (1990) Int. J. Immunopharmacol. 12:225-223;Hirata et al. (1994) Pharm. Bull. 17:739-741), sulfatedglycosaminoglycans, sulfated polysaccharide-peptidoglycan complex,sulfated alkyl malto-oligosaccharide (Katsuraya et al. (1994) CarbohydrRes. 260:51-61), amylopectin sulfate, N-acetyl-heparin (NAH)(Sigma-Aldrich), N-acetyl-de-O-sulfated-heparin (NA-de-o-SH)(Sigma-Aldrich), de-N-sulfated-heparin (De-NSH) (Sigma-Aldrich), andDe-N-sulfated-acetylated-heparin (De-NSAH) (Sigma-Aldrich).

The ability of NASPs to promote clotting and reduce bleeding is readilydetermined using various in vitro clotting assays (e.g., dPT and aPTTassays) and in vivo bleeding models (e.g. tail snip or cuticle bleedingtime determination in hemophilic mice or dogs). See, e.g., PDR Staff.Physicians' Desk Reference. 2004, Anderson et al. (1976) Thromb. Res.9:575-580; Nordfang et al. (1991) Thromb Haemost. 66:464-467; Welsch etal. (1991) Thrombosis Research 64:213-222; Broze et al. (2001) ThrombHaemost 85:747-748; Scallan et al. (2003) Blood. 102:2031-2037;Pijnappels et al. (1986) Thromb. Haemost. 55:70-73; and Giles et al.(1982) Blood 60:727-730. Clotting assays may be performed in thepresence of NASPs and one or more blood factors, procoagulants, or otherreagents. For example, one or more factors can be added, including butnot limited to, factor XI, factor XII, prekallikrein, high molecularweight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX,factor X, factor XIII, factor II, and von Willebrands factor, tissuefactor, factor VIIa, factor Va, and factor Xa, factor IXa, factor XIa,factor XIIa, and VIIIa; and/or one or more reagents, including but notlimited to, APTT reagent, thromboplastin, fibrin, TFPI, Russell's vipervenom, micronized silica particles, ellagic acid, sulfatides, andkaolin.

Examples 3-4 and FIGS. 2-3 confirm that the agents referred to herein asNASPs are truly “non-anticoagulant,” i.e. that they do not significantlyincrease clotting times over the range of concentrations studied. Suchcompounds can be used in the methods and compositions of the presentinvention provided that any anticoagulant activity that they may exhibitonly appears at concentrations significantly above the concentration atwhich they exhibit procoagulant activity. The ratio of the concentrationat which undesired anticoagulant properties occur to the concentrationat which desired procoagulant activities occur is referred to as thetherapeutic index for the NASP in question. The therapeutic index forNASPs of the present invention may be 5, 10, 30, 100, 300, 1000 or more.

D. Pharmaceutical Compositions

Optionally, the NASP compositions of the invention may further compriseone or more pharmaceutically acceptable excipients to provide apharmaceutical composition. Exemplary excipients include, withoutlimitation, carbohydrates, inorganic salts, antimicrobial agents,antioxidants, surfactants, buffers, acids, bases, and combinationsthereof. Excipients suitable for injectable compositions include water,alcohols, polyols, glycerine, vegetable oils, phospholipids, andsurfactants. A carbohydrate such as a sugar, a derivatized sugar such asan alditol, aldonic acid, an esterified sugar, and/or a sugar polymermay be present as an excipient. Specific carbohydrate excipientsinclude, for example: monosaccharides, such as fructose, maltose,galactose, glucose, D-mannose, sorbose, and the like; disaccharides,such as lactose, sucrose, trehalose, cellobiose, and the like;polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,starches, and the like; and alditols, such as mannitol, xylitol,maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol,myoinositol, and the like. The excipient can also include an inorganicsalt or buffer such as citric acid, sodium chloride, potassium chloride,sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodiumphosphate dibasic, and combinations thereof.

A composition of the invention can also include an antimicrobial agentfor preventing or deterring microbial growth. Nonlimiting examples ofantimicrobial agents suitable for the present invention includebenzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate, thimersol, and combinations thereof.

An antioxidant can be present in the composition as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe NASP or other components of the preparation. Suitable antioxidantsfor use in the present invention include, for example, ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant can be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (BASF, Mount Olive, N.J.); sorbitan esters; lipids,such as phospholipids such as lecithin and other phosphatidylcholines,phosphatidylethanolamines (although preferably not in liposomal form),fatty acids and fatty esters; steroids, such as cholesterol; chelatingagents, such as EDTA; and zinc and other such suitable cations.

Acids or bases can be present as an excipient in the composition.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The amount of the NASP (e.g., when contained in a drug delivery system)in the composition will vary depending on a number of factors, but willoptimally be a therapeutically effective dose when the composition is ina unit dosage form or container (e.g., a vial). A therapeuticallyeffective dose can be determined experimentally by repeatedadministration of increasing amounts of the composition in order todetermine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the nature and function of the excipient and particularneeds of the composition. Typically, the optimal amount of anyindividual excipient is determined through routine experimentation,i.e., by preparing compositions containing varying amounts of theexcipient (ranging from low to high), examining the stability and otherparameters, and then determining the range at which optimal performanceis attained with no significant adverse effects. Generally, however, theexcipient(s) will be present in the composition in an amount of about 1%about 99% by weight, preferably from about 5% to about 98% by weight,more preferably from about 15 to about 95% by weight of the excipient,with concentrations less than 30% by weight most preferred. Theseforegoing pharmaceutical excipients along with other excipients aredescribed in “Remington: The Science & Practice of Pharmacy”, 19th ed.,Williams & Williams, (1995), the “Physician's Desk Reference”, 52nd ed.,Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H., Handbook ofPharmaceutical Excipients, 3rd Edition, American PharmaceuticalAssociation, Washington, D.C., 2000.

The compositions encompass all types of formulations and in particularthose that are suited for injection, e.g., powders or lyophilates thatcan be reconstituted with a solvent prior to use, as well as ready forinjection solutions or suspensions, dry insoluble compositions forcombination with a vehicle prior to use, and emulsions and liquidconcentrates for dilution prior to administration. Examples of suitablediluents for reconstituting solid compositions prior to injectioninclude bacteriostatic water for injection, dextrose 5% in water,phosphate buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof. With respect to liquidpharmaceutical compositions, solutions and suspensions are envisioned.Additional preferred compositions include those for oral, ocular, orlocalized delivery.

The pharmaceutical preparations herein can also be housed in a syringe,an implantation device, or the like, depending upon the intended mode ofdelivery and use. Preferably, the NASP compositions described herein arein unit dosage form, meaning an amount of a conjugate or composition ofthe invention appropriate for a single dose, in a premeasured orpre-packaged form.

The NASP compositions herein may optionally include one or moreadditional agents, such as hemostatic agents, blood factors, or othermedications used to treat a subject for a condition or disease.Particularly preferred are compounded preparations including one or moreblood factors such as factor XI, factor XII, prekallikrein, highmolecular weight kininogen (HMWK), factor V, factor VII, factor VIII,factor IX, factor X, factor XIII, factor II, factor VIIa, and vonWillebrands factor. NASP compositions may also include otherprocoagulants, such as an activator of the intrinsic coagulationpathway, including but not limited to, factor Xa, factor IXa, factorXIa, factor XIIa, and VIIIa, prekallekrein, and high-molecular weightkininogen; or and activator of the extrinsic coagulation pathway,including but not limited to, tissue factor, factor VIIa, factor Va, andfactor Xa. NASP compositions may include naturally occurring, synthetic,or recombinant clotting factors or fragments, variants or covalentlymodified derivatives thereof that retain biological activity (i.e.,promote clotting). Alternatively, such agents can be contained in aseparate composition from the NASP and co-administered concurrently,before, or after the NASP composition of the invention.

E. Administration

At least one therapeutically effective cycle of treatment with a NASPwill be administered to a subject. By “therapeutically effective cycleof treatment” is intended a cycle of treatment that when administered,brings about a positive therapeutic response with respect to treatmentof an individual for a bleeding disorder. Of particular interest is acycle of treatment with a NASP that improves hemostasis. By “positivetherapeutic response” is intended that the individual undergoingtreatment according to the invention exhibits an improvement in one ormore symptoms of a bleeding disorder, including such improvements asshortened blood clotting times and reduced bleeding and/or reduced needfor factor replacement therapy.

In certain embodiments, multiple therapeutically effective doses ofcompositions comprising one or more NASPs and/or other therapeuticagents, such as hemostatic agents, blood factors, or other medicationswill be administered. The compositions of the present invention aretypically, although not necessarily, administered orally, via injection(subcutaneously, intravenously or intramuscularly), by infusion, orlocally. The pharmaceutical preparation can be in the form of a liquidsolution or suspension immediately prior to administration, but may alsotake another form such as a syrup, cream, ointment, tablet, capsule,powder, gel, matrix, suppository, or the like. Additional modes ofadministration are also contemplated, such as pulmonary, rectal,transdermal, transmucosal, intrathecal, pericardial, intra-arterial,intracerebral, intraocular, intraperitoneal, and so forth. Thepharmaceutical compositions comprising NASPs and other agents may beadministered using the same or different routes of administration inaccordance with any medically acceptable method known in the art.

In a particular embodiment, a composition of the invention is used forlocalized delivery of a NASP, for example, for the treatment of bleedingas a result of a lesion, injury, or surgery. The preparations accordingto the invention are also suitable for local treatment. For example, aNASP may be administered by injection at the site of bleeding or in theform of a solid, liquid, or ointment, preferably via an adhesive tape ora wound cover. Suppositories, capsules, in particulargastric-juice-resistant capsules, drops or sprays may also be used. Theparticular preparation and appropriate method of administration arechosen to target the site of bleeding.

In another embodiment, the pharmaceutical compositions comprising NASPsand/or other agents are administered prophylactically, e.g. beforeplanned surgery. Such prophylactic uses will be of particular value forsubjects with known pre-existing blood coagulation disorders.

In another embodiment of the invention, the pharmaceutical compositionscomprising NASPs and/or other agents, are in a sustained-releaseformulation, or a formulation that is administered using asustained-release device. Such devices are well known in the art, andinclude, for example, transdermal patches, and miniature implantablepumps that can provide for drug delivery over time in a continuous,steady-state fashion at a variety of doses to achieve asustained-release effect with a non-sustained-release pharmaceuticalcomposition.

The invention also provides a method for administering a conjugatecomprising a NASP as provided herein to a patient suffering from acondition that is responsive to treatment with a NASP contained in theconjugate or composition. The method comprises administering, via any ofthe herein described modes, a therapeutically effective amount of theconjugate or drug delivery system, preferably provided as part of apharmaceutical composition. The method of administering may be used totreat any condition that is responsive to treatment with a NASP. Morespecifically, the compositions herein are effective in treating bleedingdisorders, including hemophilia A, hemophilia B, von Willebrand disease,idiopathic thrombocytopenia, a deficiency of one or more contactfactors, such as Factor XI, Factor XII, prekallikrein, and highmolecular weight kininogen (HMWK), a deficiency of one or more factorsassociated with clinically significant bleeding, such as Factor V,Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II(hypoprothrombinemia), and von Willebrands factor, a vitamin Kdeficiency, a disorder of fibrinogen, including afibrinogenemia,hypofibrinogenemia, and dysfibrinogenemia, an alpha₂-antiplasmindeficiency, and excessive bleeding such as caused by liver disease,renal disease, thrombocytopenia, platelet dysfunction, hematomas,internal hemorrhage, hemarthroses, surgery, trauma, hypothermia,menstruation, and pregnancy.

Those of ordinary skill in the art will appreciate which conditions aspecific NASP can effectively treat. The actual dose to be administeredwill vary depending upon the age, weight, and general condition of thesubject as well as the severity of the condition being treated, thejudgment of the health care professional, and conjugate beingadministered. Therapeutically effective amounts can be determined bythose skilled in the art, and will be adjusted to the particularrequirements of each particular case.

Generally, a therapeutically effective amount will range from about 0.01mg/kg to 200 mg/kg of a NASP daily, more preferably from about 0.01mg/kg to 20 mg/kg daily, even more preferably from about 0.02 mg/kg to 2mg/kg daily. Preferably, such doses are in the range of 0.01-50 mg/kgfour times a day (QID), 0.01-10 mg/kg QID, 0.01-2 mg/kg QID, 0.01-0.2mg/kg QID, 0.01-50 mg/kg three times a day (TID), 0.01-10 mg/kg TID,0.01-2 mg/kg TID, 0.01-0.2 mg/kg TID, 0.01-100 mg/kg twice daily (BID),0.01-10 mg/kg BID, 0.01-2 mg/kg BID, or 0.01-0.2 mg/kg BID. The amountof compound administered will depend on the potency of the specific NASPand the magnitude or procoagulant effect desired and the route ofadministration.

A NASP (again, preferably provided as part of a pharmaceuticalpreparation) can be administered alone or in combination with otherNASPs or therapeutic agents, such as hemostatic agents, blood factors,or other medications used to treat a particular condition or diseaseaccording to a variety of dosing schedules depending on the judgment ofthe clinician, needs of the patient, and so forth. The specific dosingschedule will be known by those of ordinary skill in the art or can bedetermined experimentally using routine methods. Exemplary dosingschedules include, without limitation, administration five times a day,four times a day, three times a day, twice daily, once daily, threetimes weekly, twice weekly, once weekly, twice monthly, once monthly,and any combination thereof. Preferred compositions are those requiringdosing no more than once a day.

A NASP can be administered prior to, concurrent with, or subsequent toother agents. If provided at the same time as other agents, the NASP canbe provided in the same or in a different composition. Thus, NASPs andother agents can be presented to the individual by way of concurrenttherapy. By “concurrent therapy” is intended administration to a subjectsuch that the therapeutic effect of the combination of the substances iscaused in the subject undergoing therapy. For example, concurrenttherapy may be achieved by administering a dose of a pharmaceuticalcomposition comprising a NASP and a dose of a pharmaceutical compositioncomprising at least one other agent, such as a hemostatic agent orcoagulation factor (e.g. FVIII or FIX), which in combination comprise atherapeutically effective dose, according to a particular dosingregimen. Similarly, one or more NASPs and therapeutic agents can beadministered in at least one therapeutic dose. Administration of theseparate pharmaceutical compositions can be performed simultaneously orat different times (i.e., sequentially, in either order, on the sameday, or on different days), so long as the therapeutic effect of thecombination of these substances is caused in the subject undergoingtherapy.

F. Applications

In one aspect, NASPs may be used in the methods of the invention forimproving hemostasis in treating bleeding disorders, particularly thoseassociated with deficiencies of coagulation factors or for reversing theeffects of anticoagulants in a subject. NASPs may be administered to asubject to treat bleeding disorders, including congenital coagulationdisorders, acquired coagulation disorders, and hemorrhagic conditionsinduced by trauma. Examples of bleeding disorders that may be treatedwith NASPs include, but are not limited to, hemophilia A, hemophilia B,von Willebrand disease, idiopathic thrombocytopenia, a deficiency of oneor more contact factors, such as Factor XI, Factor XII, prekallikrein,and high molecular weight kininogen (HMWK), a deficiency of one or morefactors associated with clinically significant bleeding, such as FactorV, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor II(hypoprothrombinemia), and von Willebrands factor, a vitamin Kdeficiency, a disorder of fibrinogen, including afibrinogenemia,hypofibrinogenemia, and dysfibrinogenemia, an alpha₂-antiplasmindeficiency, and excessive bleeding such as caused by liver disease,renal disease, thrombocytopenia, platelet dysfunction, hematomas,internal hemorrhage, hemarthroses, surgery, trauma, hypothermia,menstruation, and pregnancy. In certain embodiments, NASPs are used totreat congenital coagulation disorders including hemophilia A,hemophilia B, and von Willebrands disease. In other embodiments, NASPsare used to treat acquired coagulation disorders, including deficienciesof factor VIII, von Willebrand factor, factor IX, factor V, factor XI,factor XII and factor XIII, particularly disorders caused by inhibitorsor autoimmunity against blood coagulation factors, or haemostaticdisorders caused by a disease or condition that results in reducedsynthesis of coagulation factors.

The needs of the patient will depend on the particular bleeding disorderbeing treated. For example, a NASP may be administered to treat achronic condition (e.g., a congenital or acquired coagulation factordeficiency) in multiple doses over an extended period. Alternatively, aNASP may be administered to treat an acute condition (e.g., bleedingcaused by surgery or trauma, or factor inhibitor/autoimmune episodes insubjects receiving coagulation replacement therapy) in single ormultiple doses for a relatively short period, for example one to twoweeks. In addition, NASP therapy may be used in combination with otherhemostatic agents, blood factors, and medications. For example, thesubject may be administered a therapeutically effective amount of one ormore factors selected from the group consisting of factor XI, factorXII, prekallikrein, high molecular weight kininogen (HMWK), factor V,factor VII, factor VIII, factor IX, factor X, factor XIII, factor II,factor VIIa, and von Willebrands factor. Treatment may further compriseadministering a procoagulant, such as an activator of the intrinsiccoagulation pathway, including factor Xa, factor IXa, factor XIa, factorXIIa, and VIIIa, prekallekrein, and high-molecular weight kininogen; oran activator of the extrinsic coagulation pathway, including tissuefactor, factor VIIa, factor Va, and factor Xa. In addition, transfusionof blood products may be necessary to replace blood loss in subjectsexperiencing excessive bleeding, and in cases of injury, surgical repairmay be appropriate to stop bleeding.

The invention also provides a method for reversing the effects of ananticoagulant in a subject, the method comprising administering atherapeutically effective amount of a composition comprising a NASP tothe subject. In certain embodiments, the subject may have been treatedwith an anticoagulant including, but not limited to, heparin, a coumarinderivative, such as warfarin or dicumarol, TFPI, AT III, lupusanticoagulant, nematode anticoagulant peptide (NAPc2), active-siteblocked factor VIIa (factor VIIai), factor IXa inhibitors, factor Xainhibitors, including fondaparinux, idraparinux, DX-9065a, and razaxaban(DPC906), inhibitors of factors Va and VIIIa, including activatedprotein C (APC) and soluble thrombomodulin, thrombin inhibitors,including hirudin, bivalirudin, argatroban, and ximelagatran. In certainembodiments, the anticoagulant in the subject may be an antibody thatbinds a clotting factor, including but not limited to, an antibody thatbinds to Factor V, Factor VII, Factor VIII, Factor IX, Factor X, FactorXIII, Factor II, Factor XI, Factor XII, von Willebrands factor,prekallikrein, or high molecular weight kininogen (HMWK).

In certain embodiments, a NASP can be administered alone orcoadministered with one or more different NASPs and/or in combinationwith one or more other therapeutic agents for reversing the effects ofan anticoagulant in the subject. For example, the subject may beadministered a therapeutically effective amount of a compositioncomprising a NASP and one or more factors selected from the groupconsisting of factor XI, factor XII, prekallikrein, high molecularweight kininogen (HMWK), factor V, factor VII, factor VIII, factor IX,factor X, factor XIII, factor II, factor VIIa, and von Willebrandsfactor. Treatment may further comprise administering a procoagulant,such as an activator of the intrinsic coagulation pathway, includingfactor Xa, factor IXa, factor XIa, factor XIIa, and VIIIa,prekallekrein, and high-molecular weight kininogen; or an activator ofthe extrinsic coagulation pathway, including tissue factor, factor VIIa,factor Va, and factor Xa.

In another aspect, the invention provides a method for improvingclotting in a subject undergoing a surgical or invasive procedure, themethod comprising administering a therapeutically effective amount of acomposition comprising a non-anticoagulant sulfated polysaccharide(NASP) to the subject. In certain embodiments, the NASP can beadministered alone or coadministered with one or more different NASPsand/or in combination with one or more other therapeutic agents to thesubject undergoing a surgical or invasive procedure. For example, thesubject may be administered a therapeutically effective amount of one ormore factors selected from the group consisting of factor XI, factorXII, prekallikrein, high molecular weight kininogen (HMWK), factor V,factor VII, factor VIII, factor IX, factor X, factor XIII, factor II,factor VIIa, and von Willebrands factor. Treatment may further compriseadministering a procoagulant, such as an activator of the intrinsiccoagulation pathway, including factor Xa, factor IXa, factor XIa, factorXIIa, and VIIIa, prekallekrein, and high-molecular weight kininogen; oran activator of the extrinsic coagulation pathway, including tissuefactor, factor VIIa, factor Va, and factor Xa.

In another aspect, the invention provides a method of inhibiting TFPIactivity comprising combining a composition comprising TFPI with asufficient amount of a NASP to inhibit TFPI activity. In certainembodiments, TFPI activity is inhibited in a subject by a methodcomprising administering a therapeutically effective amount of acomposition comprising a NASP to the subject. In certain embodiments,the invention provides a method of inhibiting TFPI activity in abiological sample, the method comprising combining the biological sample(e.g., blood or plasma) with a sufficient amount of a NASP to inhibitTFPI activity.

III. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Material and Methods A. Reagents

Heparin and modified heparins, and fucoidan were purchased from Sigma(St. Louis, Mo.). The source of pentosan polysulfate sodium (PPS) wasthe prescription drug Elmiron obtained from Ortho-McNeil Pharmaceuticals(Raritan, N.J.). Human plasmas were obtained from George King Biomedical(Overland Park, Kans.). Factors VIIa and human recombinant TFPI werefrom American Diagnostica (Stamford, Conn.) and Factor VIII wasprescription ReFacto® obtained from Wyeth Pharmaceuticals (Madison,N.J.). SIMPLASTIN EXCEL and APTT reagent were obtained from bioMerieux(Durham, N.C.) or Organon Teknika (Roseland, N.J.).

B. Animals

Hem-A mice (homozygous for the exon 16 FVIII KO allele) were licensedfrom John Hopkins University, and Hem-B mice (homozygous for the exon1-3 FIX KO) were licensed from University of North Carolina at ChapelHill. All animal procedures were performed according to “Guide for theCare and Use of Laboratory Animals” (National Research Council. Guidefor the care and use of laboratory animals. Washington, D.C.: NationalAcademy Press; 1996) and all procedures were reviewed and approved by aninstitutional animal care and use committee.

C. Clotting Assays

Activated Partial Thromboplastin Time (aPTT) Assay The aPTT assay wasperformed as described previously with modifications (PDR StaffPhysicians' Desk Reference. 2004, Anderson Lo, Barrowcliffe, T. W.,Holmer, E., Johnson, E. A., Sims, G. E. C. Thromb. Res. 1976;9:575-580). 25 mM CaCl₂ and fibrin cups (Fisher) were pre-warmed to 37°C. 0.1 ml of thawed human plasma (normal or hemophilic) was added towarmed test tubes. 5 μl of saline (e.g. Sigma) or 5 μl of test agent(e.g., NASP) dissolved in saline was incubated with 95 μl of plasma for30 minutes at room temperature. APTT reagent (e.g. Organon Teknika) wasreconstituted in 3 ml distilled water and 0.1 ml of the reconstitutedsolution containing the APTT reagent was added to each test tube. 0.2 mlof plasma containing the test agent or saline control and aPTT reagentwere transferred from test tubes to pre-warmed fibrin cups and incubatedfor 2-3 minutes. 0.1 ml of pre-warmed 25 mM CaCl₂ was added to initiateclotting, and the time for plasma clotting was measured with a BBLFIBROSYSTEM fibrometer.

Dilute Prothrombin Time (dPT) Assay

The dPT assay used was a modified standard clinical PT assay (Nordfanget al. (1991) Thromb Haemost 66:464-467; Welsch et al. (1991) ThrombosisResearch 64: 213-222). SIMPLASTIN EXCEL thromboplastin reagent (OrganonTeknika) was reconstituted with the manufacturer's diluent and furtherdiluted 1:100 in 0.9% saline. The thromboplastin reagent, 25 mM CaCl₂,and plasma samples were pre-warmed to 37° C. before initiating theassay. 100 μl of thawed plasma was aliquoted into microcentrifuge tubes.For measurements of inhibition of TFPI activity, 5 μl of saline (e.g.Sigma) or 5 μl of test agent (e.g. sulfated polysaccharide) was added to95 μl of plasma and incubated for approximately 30 minutes at roomtemperature. 100 μl of the diluted thromboplastin reagent and 100 μl of25 mM CaCl₂ were added to fibrin cups (Fisher) prewarmed to 37° C. 100μl of plasma (normal or hemophilic) containing the test agent or salinecontrol was added to the fibrin cups containing the thromboplastinreagent and CaCl₂ to initiate clotting. The time for plasma clotting wasmeasured with a BBL FIBROSYSTEM fibrometer.

Animal Bleeding Time Assays

The bleeding time assay can be used to measure changes in hemostasisfunction in normal or hemophilic (FVIII or FIX or vWF deficient) rodentsfollowing administration of a test agent (e.g., vehicle control orNASP). A test agent (e.g., vehicle control or NASP) is administered to arodent once or twice daily orally, parenterally, or by continuousinfusion. For example, 0.1 ml/10 g body weight (subscapular) of a testagent at a dose ranging from 0.1 to 10 mg/kg can be administered withsmall gauge needles twice a day for at least one day and preferably morethan 3 days. On the day bleeding time is assayed, rodents areanesthetized with ketamine/xylazine (or isoflurane). Rodents are linedup on a sterile pad with a petri dish of saline for tail immersion. EMLAcreme is applied to the tail of rodents at an intended cut site. Formice, the very tip of the tail is snipped, and the tail is placed intothe saline dish and a counter is started. For rats, an 8 mm long by 1 mmdeep incision is made on the dorsal part of the rat tail, which is thentransferred into saline. The time for cessation of visible bleeding intothe saline is recorded. For rodents, bleeding times are approximately 10minutes for normal control mice and 6 minutes for normal control rats.After completion of the bleeding time assay, the rodent's tail is driedwith sterile gauge, verified for hemostasis, and the rodent is returnedto the cage. Silver nitrate can be applied to the cut site if necessary.

Alternatively, bleeding times can be measured in mice (Broze et al.(2001) Thromb. Haemost. 85:747-748) or in dogs (Scallan et al. (2003)Blood 102:2031-2037; Pijnappels et al. (1986) Thromb. Haemost. 55:70-73)by other methods. Alternative or additional pharmacodynamic endpointsmay include sampling of blood from NASP-treated subjects for directanalysis or for plasma isolation, and measurement of ex vivo clottingtimes (e.g., Whole Blood Clotting Time and/or PT and/or APTT) orcoagulation factor levels.

Whole Blood Clotting Time (WBCT) Assay

The WBCT assay was performed as follows. Mice were briefly anesthetizedin an isoflurane chamber. The mice were then bled (e.g. 150 μl) from theretro orbital plexus into plastic blood collection tubes. The tubes wereplaced in a 37° C. water bath and a stop watch was used to measureclotting time. During this period, the tubes were inverted at 1 minuteintervals. The time required for blood clotting (full/not partial clot)was measured.

Statistical Analyses

For the clotting assays, the Student's t-test was used to analyze thesignificance between NASP-treated samples and vehicle controls. Datafrom mouse bleeding tests were studied for significance from vehiclecontrols (or other groups as indicated in the tables below) by one-wayChi-squared analysis. Nearly identical results were obtained by Fisher'sexact test.

Example 2 TFPI Increases Clotting Time in dPT Assay

The following experiments were performed to demonstrate that TFPIincreases clotting time in the dPT assay and to determine a TFPIconcentration for use in subsequent NASP experiments. A 100 μg/mL TFPIstock solution (American Diagnostica, Stamford, Conn.) was sequentiallydiluted in saline to generate TFPI solutions at the followingconcentrations: 20, 15, 10, 6, and 2 μg/mL. 5 μl of these TFPI dilutionswere mixed with 95 μl of FVIII deficient plasma and incubated at roomtemperature for 30 minutes. dPT assays were performed as follows:SIMPLASTIN thromboplastin was diluted 1:100 in saline and prewarmed to37° C. 25 mM CaCl₂ and 100 μl of test plasma containing TFPI wasprewarmed to 37° C. 100 μl SIMPLASTIN thromboplastin and 100 μl of CaCl₂were mixed and clotting time was measured using a BBL fibrometer. Theresults are summarized in Table 1.

TABLE 1 Clotting Times in Presence of TFPI TFPI concentration Clottingtime in plasma (μg/mL) (seconds) 1 >200 0.75 173 0.5 98 0.3 94 0.1 60TFPI increased the clotting time of Hem-A plasma with a linear doseresponse (see FIG. 1). Based on these data, a concentration of 0.5 μg/mlTFPI was chosen for assays of NASP procoagulant function.

Example 3 Screening for NASPs

Sulfated polysaccharide compounds, including modified heparins, pentosanpolysulfate, and fucoidan were tested for anti-coagulant activity andcompared to heparin to determine whether they qualified as“non-anticoagulants.” The compounds tested are listed in Table 2.

TABLE 2 NASPs Tested for Anti-Coagulant Activity NASP Company/Cat. # MW(kd) N-Acetyl-Heparin Sigma Chem. Co. 18 (NAH) A8036 N-Acetyl-de-O-Sigma Chem. Co. 18 Sulfated-Heparin A6039 (NA-de-o-SH) De-N-Sulfated-Sigma Chem. Co. 18 Heparin (De-NSH) D4776 De-N-Sulfated- Sigma Chem. Co.18 Acetylated-Heparin D9808 (De-NSAH) Pentosan Ivax 5 PolysulphatePharmaceuticals, Sodium (PPS) Inc. NDC 17314-9300-1 Fucoidan Sigma Chem.Co. 100 F5631 Sodium Heparin Sigma Chem. Co. 18 H4784

Test compounds were diluted to 100 μM, 10 μM, 2 μM and 200 nM. For eachtest compound, 12.5 μl of a diluted solution containing the testcompound was added to 237.5 μl of Hem-A plasma and incubated at roomtemperature. 100 μl of plasma containing the test compound was removedfor dPT assays of plasma clotting time as described in Example 2. Theresults are summarized in Table 3 below.

TABLE 3 Effect of NASPs on Clotting Time* According to dPT Assay NASPConcentration NA-de- De- (nM) NAH O-SH De-N-SH N—S-AH PPS HeparinFucoidan 10 38 40 40 39 39 40 39 100 37 40 38 37 37 92 38 500 36 40 3840 40 400 60 5000 38 40 41 55 70 *The values shown are clotting times(seconds) for selected polysaccharides. The clotting time of Hem-Aplasma in the absence of NASPs is 41.5 seconds.

As shown in Table 3 and FIG. 2, heparin at concentrations exceeding 10nM was markedly anticoagulant whereas N-acetyl heparin (NAH),N-acetyl-de-O-sulfated heparin (NA-de-O-SH), de-N-sulfated heparin(De-N-SH) showed little or no prolongation of clotting time atconcentrations>5000 nM. Likewise, fucoidan and PPS were only weaklyanticoagulant, exhibiting 50% prolongation of clotting time atconcentrations approximately 10- to 100-fold higher, respectively, thanheparin and are hence denoted “non-anticoagulant.” A nearly identicalprofile was observed with normal human plasma (data not shown).

Example 4 Effect of NASPs on Clotting of Human Plasma According to aPTTAssay

The effect of NASPs on the clotting time of plasma was also measuredusing an aPTT assay to determine whether they qualify as“non-anticoagulants.” Dilutions of FACT, a “normal” human referenceplasma (George King Biomedical), were made in human Hem-A plasma togenerate plasma with concentrations of normal plasma from 0.31-100%. TheaPTT assay was then performed as follows: 100 μl of a FACT-Hem-A plasmamixture and 100 μl of aPTT reagent were mixed and incubated at 37° C.for 3 minutes. 100 μA of CaCl₂ was added, and the time for plasmaclotting was measured using a BBL fibrometer. The results are shown inTable 4.

TABLE 4 Effect of FACT Concentration on Clotting Time FACT conc. inHem-A aPTT time plasma (%) (seconds) 100 40 50 40 25 42 10 50 5 54 2.560 1.25 64 0.63 69 0.31 76 0 96

Based on this data, a FACT concentration of 1.25% was chosen for assaysscreening NASPs for procoagulant activity. The effect of NASPs on theclotting time of plasma was determined as follows: 5 μl of a NASP wasadded to 95 μl of 1.25% FACT diluted in human Hem-A plasma and incubatedat room temperature for 30 minutes. aPTT assays were performed todetermine plasma clotting time as described in Example 1. The resultsare shown in Table 5.

TABLE 5 Effect of NASPs on Plasma Clotting Time According to the aPTTAssay NASP Con- NAH PPS Fucoidan Heparin centration clotting clottingclotting clotting (nM) time (sec) time (sec) time (sec) time (sec) 0.1671 0.8 70 70 70 70 4 69 71 71 70 20 67 72 75 200 100 74 80 119 Notclotted 500 85 113 Not clotted

Further validation of “NASP” activity was demonstrated by evaluation ofthree compounds in an APTT clotting assay with Hem-A plasma.Concentrations producing approximately 50% prolongation in clotting timewere 10- or 100- or >500-fold higher for fucoidan, PPS, and NAH,respectively, than for heparin (see FIG. 3).

Example 5 Inhibition of TFPI Activity by NASPs

A. Preincubation of TFPI with NASPs Prior to Addition to Plasma

Inhibition of TFPI activity by NASPs was assessed in dPT clotting assayswith normal or hemophilic plasma and added recombinant TFPI. Dilutedrecombinant TFPI was preincubated with NASPs for 5 minutes at roomtemperature before plasma was added. After addition of plasma, themixture was incubated for an additional 25 minutes followed by dPTinitiation. The results for assays performed in Hem-A plasma are shownin Table 6 and FIG. 4.

TABLE 6 NASP Inhibition of TFPI Activity in Hem-A Plasma NASP FucoidanPPS NAH Concen- clotting clotting clotting tration (nM) time (sec) time(sec) time (sec) 500 75 74 84 100 46 57 99 20 54 55 141 4 72 91 160 0.8108 111 158 0.16 130 158 144 Clotting time of Hem-A plasma alone is 44seconds. Clotting time of Hem-A plasma + TFPI is 151 seconds.

TFPI at a final concentration of approximately 0.5 μg/ml prolonged theclotting time of plasma from approximately 40 seconds to 100-200 secondsdepending on the experiment and source of human plasma. If TFPI activitywere inhibited by sulfated polysaccharides, then a shortening ofclotting time should be observed in the presence of NASPs (see Nordfanget al. (1991) Thromb. Haemost. 66(4):464-467). As shown in FIG. 4,addition of fucoidan and PPS at concentrations greater than 1 nMsignificantly accelerated clotting of Hem-A plasma containing TFPI. Incontrast, NAH required concentrations of approximately 100 nM to shortenclotting time, and heparin (not shown) only prolonged clotting times.Importantly, at optimal concentrations of PPS or fucoidan, the clottingtime was shortened to the no TFPI, or vehicle control levels, orslightly below, and the breadth of neutralization of TFPI effect spannedat least a 100-fold range (e.g., 5 to 500 nM).

The acceleration of plasma clotting by the NASPs in the presence of TFPIwas also tested in Hem-B and normal plasma. The results for assaysperformed in Hem-B plasma are shown in Table 7′ and FIG. 5.

TABLE 7 NASP Inhibition of TFPI Activity in Hem-B Plasma NASP FucoidanPPS NAH Concen- clotting clotting clotting tration (nM) time (sec) time(sec) time (sec) 500 60 56 68 100 50 52 94 20 54 65 106 4 80 82 106 0.895 90 101 0.16 108 106 102 Clotting time of Hem-B alone, no TFPI: 46seconds. Clotting time of Hem-B + TFPI: 101 seconds.

The acceleration of plasma clotting by the NASPs, presumably byinhibition of TFPI activity, was similarly demonstrated in Hem B plasma(Table 7 and FIG. 5) and normal human plasma (data not shown). The rankorder of potency between NASPs was identical to the studies with Hem Aplasma and the concentration-response profile was nearly identical.

B. Inhibition of TFPI Activity with No Preincubation of TFPI with NASPs

Experiments were repeated without a preincubation of the sulfatedpolysaccharides with TFPI prior to exposure to plasma. To extend thestringency of the test for NASP inhibition of TFPI activity, TFPI wasadded to the plasma before the NASP was added. The results are shown inTable 8 and FIG. 6.

TABLE 8 Inhibition of TFPI by NAH and PPS in Hem-A Plasma WithoutPreincubation NASP NAH PPS Fucoidan Concen- clotting clotting clottingtration (nM) time (sec) time (sec) time (sec) 500 89 73 90 100 125 76 5420 184 81 59 4 180 156 78 0.8 165 192 210 HemA + TFPI: 183 seconds Hem-Aalone, no TFPI: 45 seconds

As depicted in FIG. 6, the NASPs clearly demonstrated the same propertyof clotting time acceleration in Hem A plasma with nearly identicaldose-response profiles as in the preincubation studies (FIG. 4).Interestingly, fucoidan was most potent and the concentration window forsignificant clotting acceleration was greater than 100-fold. Thesestudies therefore established that certain NASPs such as PPS andfucoidan could exhibit TFPI neutralizing activity, and that suchefficacy was demonstrated across a very broad range of concentrationswherein net anticoagulation was not observed.

Example 6 Improvement in Hemophilic Plasma Coagulation by NASPs in theAbsence of TFPI Supplementation

The ability of NASPs to accelerate clotting of factor-deficient plasmain the absence of TFPI supplementation was also tested in dPT assays. Aprocoagulant response, if observed, may be related to neutralization ofendogenous TFPI activity, which is present in human plasma atapproximately 100 ng/ml (Nordfang et al., supra), largely associatedwith lipoprotein or platelets (Broze et al. (1992) Semin. Hematol.29:159-169; Broze et al. (2003) J. Thromb. Haemost. 1:1671-1675).

A. Acceleration of Clotting in Hem-A Plasma in Absence of Exogenous TFPI

The ability of NASPs to accelerate clotting of Hem-A plasma in theabsence of exogenous TFPI was tested. Fucoidan or PPS were titrated intoHem A plasma and dPT assays were performed. Additionally, thedose-response to factor VIIa was analyzed as a positive control foramplifying extrinsic pathway activation. The results are shown in FIG. 7and Table 9.

TABLE 9 Acceleration of Hem-A Plasma Clotting In Absence of ExogenousTFPI NASP Fucoidan PPS FVIIa Concen- clotting clotting clotting tration(nM) time (sec) time (sec) time (sec) 100 56 60 20 55 62 49 4 63 66 560.8 68 68 62 0.16 70 69 68 Clotting time of Hem-A alone, no NASP: 69seconds

Fucoidan and PPS both significantly accelerated the clotting time in adose-dependent fashion with fucoidan exhibiting the best potency andmaximal efficacy. As in other studies, there was a window ofprocoagulant effect that, in the case of fucoidan, ranged fromapproximately 5 nM to >100 nM. Note in FIG. 7 that while the responsecurve begins to deflect upwards at concentrations of fucoidan>100 nM,clotting is still accelerated relative to the vehicle control, andfucoidan is hence procoagulant. While the shortening of clotting timefrom about 70 seconds to 55 seconds at 20 nM fucoidan is not a largemargin, NAH had no activity. Such acceleration has been observedpreviously with procoagulant factors like FVIIa and thrombin.Accordingly, FVIIa addition to 20 nM accelerated clotting times byapproximately 20 seconds, which was greater than that of fucoidan (FIG.7). However, it is interesting to note that 20 nM fucoidan performedcomparably to a pharmacological concentration of 5 nM FVIIa.

B. Acceleration of Clotting in Hem-B plasma and FVII-Deficient Plasma inAbsence of Exogenous TFPI

Evaluation of the apparent procoagulant activity of NASPs was extendedto other human bleeding disorders by testing NASP activity in Hem Bplasma and FVII-deficient plasma. Similar results to those shown forHem-A plasma were observed for Hem-B plasma (data not shown).

Regulation of clotting in Factor VII-deficient plasma was also evaluatedin dPT assays. As expected, FVII-deficient plasma failed to clot within300 seconds without FVIIa reconstitution. Addition of FVIIa toapproximately 0.1 nM restored the clotting time to about 150 seconds(data not shown). Such a variation in clotting time shown in the dPTassay mimics some forms of human factor VII-deficiency. Titration offucoidan and PPS into FVII-deficient plasma accelerated clotting times.The results are shown in FIG. 8 and Table 10.

TABLE 10 Acceleration of Factor VII-Deficient Plasma Clotting In Absenceof Exogenous TFPI NASP Fucoidan PPS Concen- clotting clotting tration(nM) time (sec) time (sec) 500 111 113 100 74 142 20 120 159 4 147 1810.8 168 198 Clotting time with no NASP, no FVIIa: >300 seconds Clottingtime with no NASP, + 0.1 nM FVIIa: 173 seconds

As shown in FIG. 8, titration of fucoidan and PPS accelerated clottingof Factor VII-deficient plasma and, as observed with Hem A plasma,fucoidan was significantly more potent and effective than PPS. Onceagain, the therapeutic window was broad; in the case of fucoidan,substantial acceleration of clotting was observed with concentrationsranging from approximately 10 nM to 500 nM.

Example 7 Improved Hemostasis of NASP-Treated Mice

Hem A or Hem B mice were treated with PPS and fucoidan to assesspotential improvement of hemostasis in vivo. NASPs were injectedsubcutaneously as frequent dosing is reasonably well tolerated inhemophilic mice and bioavailability from this route for various sulfatedpolysaccharides has been previously established (MacGregor et al. (1985)Thromb. Haemost. 53:411-414; Millet et al. (1999) Thromb. Haemost.81:391-395). PPS and fucoidan half-lives may be as short as 1-2 hours.Therefore, a twice daily dosing regimen was adopted. Initial studiesindicated that dosing for several days was preferred over 1-2 days.

The effects of NASP treatment on coagulation regulation in the treatedmice was evaluated based on several potential endpoints, includingplasma isolation for dPT assays, blood sampling for whole blood clottingtime (WBCT) assays, acute bleeding times, and longer-term survivalfollowing tail snip or transverse incision (Broze et al. (2001) Thromb.Haemost. 85:747-748). The results from 5-day in vivo studies with PPSand fucoidan are summarized in Tables 11-13.

A. PPS Efficacy in Hem-A and Hem-B Mice The efficacy of PPS in improvingclotting in Hem-A and Hem-B mice was tested. Hem-A and Hem-B male orfemale mice were administered PPS at a dose of 0.02, 0.06, or 0.2 mg/kgor saline vehicle subcutaneously twice daily for 5 days. On the morningof the fifth day after dosing, the tail was clipped 1 cm from the tip,and behavior and survival were monitored for the next 20-24 hours. Theresults are shown in Table 11.

TABLE 11 Improved Hemostasis in PPS-Treated Hemophilic Mice Treatment %Survival Hemophilia Group n/group (20 hours post-cut) A(FVIII-deficient) Vehicle control 8 25   0.02 mg/kg 5 20   0.06 mg/kg 944 ^(#)  0.2 mg/kg 5 40   B (FIX-deficient) Vehicle control 8 25   0.06mg/kg 9 44 ^(#) Mice were randomized and dosed subcutaneously withindicated agent twice daily for 4.5 days followed by tail cut (t = 0).^(#) p = 0.07 vs. vehicle

Treatment of Hem-A mice with PPS at 0.06 mg/kg showed a nearly two-foldimprovement in survival, but the result was not statisticallysignificant (0.05<p<0.1) (Table 11). Therapeutic, benefit was furthersupported by visual observations by technical staff blinded to treatmentgroup who observed more normal behavior (less lethargy and hunching) andless extensive bleeding in the mid and high dose animals relative to thevehicle controls. Likewise, subsequent treatment of Hem-B mice with themore effective dose of 0.06 mg/kg subcutaneously twice daily yielded anidentical result as that observed in the FVIII-deficient mice.

The efficacy of PPS in improving clotting in Hem-B mice was furthertested in dPT assays. All mice were bled prior to the study to establishbaseline (pretest) clotting times. Mice (14 weeks old) were treatedsubcutaneously twice a day with PPS for 4.5 days at the following doses:2, 0.3 and 0.06 mg/kg in a volume of 250 Mice were bled after 4.5 days,and clotting times were determined from collected blood samples. Theresults are shown in Table 12.

TABLE 12 Clotting for Hem-B Mice Treated with PPS NASPs Improve dPTGroup Individual clotting times Mean clotting time (mg/kg) at 4.5 days(min) at 4.5 days (min) 0.06 44 37 42 26 0.3 43 38 31 39 2.0 42 44 45 44Naïve Hem-B have dPT ranging from 44-50 seconds.

B. Fucoidan Efficacy in Hem-A Mice

Given the improved potency and magnitude of efficacy of fucoidanrelative to PPS in some of the clotting assays described above,additional studies were performed in Hem-A mice with fucoidan. In thefirst study with fucoidan, nearly the same regimen as described for PPSwas adopted, but with slightly different dose levels. Hem-A male micewere administered fucoidan at a dose of 0.1 or 1.0 mg/kg or salinesubcutaneously twice daily for 4 days. On the morning of the 5th day,mice received a doubled dose of fucoidan prior to the bleeding test.Survival and animal behavior were evaluated for mice treated withfucoidan compared to vehicle controls.

In a second study with fucoidan, combination therapy potential withfactor VIII was evaluated. This study was performed as described above,except on the morning of the fifth day, mice received an intravenousbolus dose of 53 mU/mouse FVIII (about 1.25% of the normal level ofFVIII) in a tail vein far up near the body. As before, the lateral tailvein, and not the artery, was transected 2 hours later at the regioncorresponding to a diameter of about 2.7 mm. In these fucoidan studies,the tail vein transection modification was utilized as it was found tomore accurately assess hemostasis and its regulation (Broze et al.(2001) Thromb. Haemost. 85:747-748). Survival and clinical observationswere recorded for 20-24 hours. The results are shown in Table 13.

TABLE 13 Efficacy of Fucoidan and Combination Fucoidan + FVIII inHemophila A Mice % Survival Treatment Group n/group 9 hr 20 hr Vehiclecontrol 14 21    7   Fucoidan 13 61 * 38 ⁺  (0.1 mg/kg) Factor VIII  757 * 57 *  (1.25% reconstitution) Fucoidan + FVIII  7 86 * 86 *^(#) Micewere randomized and dosed subcutaneously with vehicle or NASP twicedaily for 4.5 days followed by tail vein incision (t = 0). Whereindicated, FVIII was administered 2 hours prior to tail cut. Note that1% FVIII reconstitution yields ~10% survival whereas 2% FVIIIreconstitution provides ~100% survival in these mice. * p < 0.05 vs.vehicle ⁺ p = 0.06 vs. vehicle ^(#) p = 0.06 vs. fucoidan

In the first study, treatment of mice with fucoidan at a dose of 0.1mg/kg appeared to be more efficacious than treatment at a dose of 1.0mg/kg (survival at about 10 hours was 1/6 for vehicle, 4/6 for 0.1mg/kg, and 3/6 for 1.0 mg/kg). Hence, the second study was performedwith fucoidan at a dose of 0.1 mg/kg.

As indicated in the top two rows of Table 13, fucoidan treatment of HemA mice significantly improved bleeding survival. Animal behavior, asdescribed above, was more normal in all the fucoidan-treated mice duringthe first 8-10 hours post-incision, and was clearly improved long-termin nearly half the animals.

Combination therapy potential was preliminarily assessed by treatingmice with FVIII+/−fucoidan (Table 13). A preliminary dose-guiding studywith FVIII administration alone to Hem A mice two hours prior to tailincision indicated a very steep dose-response relationship for survival.ReFacto® administration to 1% of normal yielded about 10% survival,whereas dosing to 2% of normal yielded about 100% survival (data notshown). Accordingly, a dose of 1.25% FVIII reconstitution was selectedto give approximately 50% survival. Notably, the percent survival in thefucoidan+FVIII treatment group was consistently higher than eitherfucoidan or FVIII alone. Thus, the results of the PPS and fucoidanstudies indicate that hemostasis is improved in animals models ofhemophilia following select NASP administration.

CONCLUSION

A series of studies were undertaken to test NASPs for improvement ofclotting in ex vivo and in vivo hemophilia models. Sulfatedpolysaccharides were identified with substantially reduced anticoagulantproperties relative to heparin. A subset of those NASPs, namely fucoidanand PPS, were shown to potently inhibit the activity of TFPI, thepredominant downregulator of the extrinsic pathway of blood coagulation.Fucoidan and PPS improved the dilute prothrombin clotting times of humanplasma deficient in factors VII, VIII, or IX. Therapeutic benefit offucoidan or PPS treatment in vivo was apparent from bleeding tests ofhemophilic mice.

Both PPS and fucoidan may exhibit anticoagulant activity at higherconcentrations, likely as a result of heparin cofactor II interaction(Church et al. (1989) J. Biol. Chem. 264:3618-3623; Giedrojc et al.(1999) J. Cardiovasc. Pharmacol. 34:340-345). PPS administeredsubcutaneously to rats requires doses>5 mg/kg to prolong clotting(Giedrojc et al., supra), and fucoidan seems well-tolerated in rabbitseven when given intravenously at 10 mg/kg (Granert et al. (1999) Infect.Immun. 67:2071-2074). Hence, the current results show that hemostasis isimproved at doses≦0.1 mg/kg in hemophilic rodents. Dose levels thatimproved hemostasis in vivo were lower than those causing other reportedeffects (Toida et al. (2003) Trends in Glycoscience and Glycotechnology15:29-46; Luyt et al. (2003) J. Pharmacol. Exp. Ther. 305:24-30; Berteauet al. (2003) Glycobiologyl3:29 R-40R; Granert et al., supra; andSweeney et al. (2002) Blood 99:44-51).

Without being bound by a particular theory, NASP inhibition of TFPI mayaccount in part for the observed improvements in coagulation ex vivo andin vivo. Neutralization of TFPI by antibodies has been shown to improvehemostasis in a rabbit Hem A model and to accelerate clotting of humanhemophilic plasma (Nordfang et al., supra; Welsch et al., supra; andErhardtsen et al. (1995) Blood Coagul. Fibrinolysis 6:388-394). In thecurrent studies, only compounds inhibiting TFPI activity also reducedclotting times in the hemophilic plasma dPT assays. Additionally,fucoidan exhibited better potency and perhaps greater maximal effectcompared to PPS in the dPT clotting test when the TFPI was first mixedinto plasma to best mimic the natural setting. Likewise, fucoidantreatment of mice yielded somewhat better efficacy than PPS althoughundefined relative pharmacokinetics may have, influenced the bleedingoutcomes.

It is noteworthy that such behavior was not apparent with all testedNASPs. For example, NAH exhibited only weak TFPI-neutralizing activity(FIGS. 4-6) and did not accelerate hemophilic plasma clotting times inthe absence of TFPI addition (data not shown). Moreover, three NASPswhich failed to show inherent anticoagulant activity at concentrationsup to 5000 nM (FIG. 2; De-N-S-AH, De-N-SH, and NA-De-O-SH) did notexhibit any TFPI-neutralizing activity and likewise failed to accelerateclotting times in Hem A plasma (data not shown).

The magnitude of improved hemostasis observed with NASPs appears to beclinically relevant. Improved clotting times of Hem A plasma at optimalfucoidan concentrations were comparable to FVIIa supplementation atapproximately 5 nM (Example 6) which has proven effective in normalizinghemostasis in patients (Bishop et al. (2004) Nat. Rev. Drug Discov.3:684-694; Carcao et al. (2004) Blood Rev. 18:101-113; Roberts et al.(2004) Anesthesiology 100:722-730; Lee et al. (2004) Int. Anesthesiol.Clin. 42:59-76; and Brummel et al. (2004) J. Thromb. Haemost.2:1735-1744). In addition, survival benefit with NASP treatment in micewas significant (Example 7). Fucoidan acceleration of clotting in dPTassays is more pronounced with human hemophilic plasma than mouse plasma(data not shown).

An obvious consideration regarding potential clinical development of aNASP for bleeding disorders would be therapeutic index. Specifically,index between improved hemostasis and the transition toanti-coagulation. From the clotting assay results for compounds such asPPS or fucoidan in human plasma, the margin between anti-TFPI oraccelerated dPT clotting “activity” and loss of such efficacy and onsetof net anticoagulation would appear to be ≧50-fold. As mentioned abovefor the mouse studies, the index would appear in mice to be at leastten-fold. Furthermore, as a class, heparin-like sulfated polysaccharidesare generally well-tolerated.

In summary, systemic administration of select NASPs may represent aunique approach for regulating hemostasis in bleeding disorders.Pentosan polysulfate and fucoidan, in particular, inhibited TFPIactivity and improved clotting of human factor VII-, VIII-, andDC-deficient plasmas. Thus, NASP treatment improved hemostasis and mayrepresent a relatively low-cost, safe, and convenient alternative orsupplement to current coagulation factor therapies.

While the preferred embodiments of the invention have been illustratedand described, it will be appreciated that various changes, can be madetherein without departing from the spirit and scope of the invention.

1-26. (canceled)
 27. A method of identifying a sulfated polysaccharidecapable of accelerating clotting, the method comprising: measuringclotting time of a first plasma sample; contacting a second plasmasample with a composition comprising a sulfated polysaccharide;measuring the clotting time of the second plasma sample contacted withthe composition comprising a sulfated polysaccharide; and comparing theclotting time of the first plasma sample with the clotting time of thesecond plasma sample, wherein a decrease in the clotting time of thesecond plasma sample compared to the first plasma sample indicates thatthe sulfated polysaccharide accelerates clotting.
 28. The methodaccording to claim 27, wherein the sulfated polysaccharide is selectedfrom the group consisting of PPS, NAH and Fucoidan.
 29. The methodaccording to 28, wherein the sulfated polysaccharide is Fucoidan. 30.The method according to claim 27, wherein the sulfated polysaccharidehas a concentration of about 10 nM to about 5000 nM.
 31. The methodaccording to claim 27, wherein the first plasma sample and the secondplasma sample are Hem-A plasmas.
 32. The method according to claim 27,wherein the first plasma sample and the second plasma sample areFACT-diluted Hem-A plasmas.
 33. The method according to claim 32,wherein the sulfated polysaccharide is selected from the groupconsisting of PPS, NAH and Fucoidan.
 34. The method according to claim33, wherein the sulfated polysaccharide is Fucoidan.
 35. The methodaccording to claim 33, wherein the sulfated polysaccharide has aconcentration of about 0.16 nM to about 500 nM.
 36. A method ofidentifying a TFP I-inhibiting compound which is capable of enhancingblood coagulation, the method comprising: contacting a first plasmasample with a composition comprising a sulfated polysaccharide;measuring clotting time of the first plasma sample; contacting a secondplasma sample with exogenous TFPI and a composition comprising thesulfated polysaccharide; measuring clotting time in the second plasmasample; and comparing the clotting time of the first plasma sample withthe clotting time of the second plasma sample, wherein an equivalent orreduction in clotting time of the second plasma sample compared to thefirst plasma sample indicates that the sulfated polysaccharide is aTFPI-inhibiting compound.
 37. The method according to claim 36, whereinthe sulfated polysaccharide is selected from the group consisting ofPPS, NAH and Fucoidan.
 38. The method according to claim 37, wherein thesulfated polysaccharide is Fucoidan.
 39. The method according to claim37, wherein the sulfated polysaccharide has a concentration of about 5nM to about 500 nM.
 40. The method according to claim 36, wherein thefirst plasma sample and the second plasma sample are Hem-A plasmas. 41.The method according to claim 36, wherein the first plasma sample andthe second plasma sample are Hem-B plasmas.
 42. The method according toclaim 36, wherein exogenous TFPI and the composition comprising asulfated polysaccharide are preincubated with each other beforecontacting with the second plasma sample.
 43. The method according toclaim 42, wherein exogenous TFPI and the composition comprising asulfated polysaccharide are preincubated with each other for 5 minutesbefore contacting with the second plasma sample.
 44. A method ofprophylactically treating a subject diagnosed as having a bloodcoagulation disorder, the method comprising subcutaneously administeringa daily dose of a Fucoidan to the subject in combination with FactorVIII before surgery.
 45. The method according to claim 44, wherein theFucoidan is administered in a dose of about 0.1 mg/kg to about 1.0mg/kg.
 46. The method according to claim 44, wherein the daily dose isabout 0.1 mg/kg.
 47. The method according to claim 44, wherein FactorVIII is administered at a dose of about 53 mU.
 48. The method accordingto claim 44, wherein the Fucoidan is administered in combination withFactor VIII for 4 days before surgery.
 49. The method according to claim44, wherein the blood coagulation disorder is Hemophilia-A.
 50. A methodof identifying a sulfated polysaccharide capable of FVII-independentacceleration of blood clotting, the method comprising: measuringclotting time of a Factor VII-deficient plasma sample; contacting theFactor VII-deficient plasma sample with a sulfated polysaccharide;measuring clotting time of the Factor VII-deficient plasma sample aftercontacting with the sulfated polysaccharide; and comparing the clottingtime before contacting the Factor VII-deficient plasma sample with thesulfated polysaccharide with the clotting time after contacting theFactor VII-deficient plasma sample with the sulfated polysaccharide,wherein a decrease in clotting time after contacting the FactorVII-deficient plasma sample with the sulfated polysaccharide compared tobefore contacting the Factor VII-deficient plasma sample with thesulfated polysaccharide indicates that the sulfated polysaccharide iscapable of FVII-independent acceleration of blood clotting.
 51. Themethod according to claim 50, wherein the method further comprises:contacting a Factor VII-deficient plasma sample with Factor VIIa;measuring clotting time of the Factor VII-deficient plasma sample aftercontacting with Factor VIIa; and comparing the clotting time aftercontacting the Factor VII-deficient plasma sample with Factor VIIa withthe clotting time after contacting with the sulfated polysaccharide,wherein a decrease or an equal clotting time after contacting with thesulfated polysaccharide compared to the clotting time after contactingwith Factor VIIa indicates that the sulfated polysaccharide is capableof FVII-independent acceleration of blood clotting.
 52. The methodaccording to claim 50, wherein the sulfated polysaccharide is PPS orFucoidan.
 53. The method according to claim 50, wherein the sulfatedpolysaccharide is Fucoidan.
 54. The method according to claim 50,wherein the decrease in clotting time is a two-fold or more decrease inclotting time.
 55. The method according to claim 50, wherein thesulfated polysaccharide has a concentration of about 10 nM to about 500nM.